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PubMed1994

HPLC analysis of oligosaccharides in urine from oligosaccharidosis patients.

Analysis of urinary oligosaccharides by thin-layer chromatography (TLC) is used as screening procedure for 10 different lysosomal diseases. We tested the usefulness of HPLC in screening, using a CarboPac PA1 column (Dionex), pulsed amperometric detection (PAD), and post-column derivatization (PCD). Patterns from six types of oligosaccharidoses were compared with normal urinary patterns and with the TLC patterns. PAD appeared to be nonspecific and therefore is applicable only to desalted urine samples. PCD was more specific and applicable to nondesalted urine samples, albeit with a lower resolving power. Peaks in urines from oligosaccharidoses patients were identified on the basis of retention times of commercially available oligosaccharides or TLC bands after isolation and HPLC of the corresponding oligosaccharides. Abnormal oligosaccharide peaks were seen in urines from patients with alpha-mannosidosis, GM1-gangliosidosis (juvenile), GM2-gangliosidosis (Sandhoff disease), Pompe disease, and beta-mannosidosis. HPLC detected no abnormal oligosaccharides in urine from patients with fucosidosis. Although TLC is a simple and reliable screening procedure for detecting classical lysosomal diseases with oligosaccharide excretion, HPLC, by its higher resolution and possibility of quantification, can more generally be used for recognition of abnormal oligosaccharides or detection of increased excretion or content for known oligosaccharides in urine, other body fluids, and cells.

Adolescent↗
PubMed1976

Biosynthesis of chondroitin sulfate. Independent addition of glucuronic acid and N-acetylgalactosamine to oligosaccharides.

Pentasaccharide 6-sulfate and hexasaccharide 6-sulfate were prepared from chondroitin 6-sulfate. Each oligosaccharide was incubated with a chick cartilage microsomal enzyme preparation and UDP [14C] glucuronic acid and/or UDP-N-[3H] acetylgalactosamine. As previously reported by other investigators, a single sugar was added from UDP-[14C] glucuronic acid to the nonreducing end of pentasaccharide 6-sulfate and from UDP-N-[3H] acetylgalactosamine to the nonreducing end of hexasaccharide 6-sulfate. The labeled oligosaccharides were characterized by gel chromatography and degradation by chondroitinase ABC followed by identification of products. The oligosaccharides in concentrations above their Km inhibited chondroitin synthesis on endogenous primers, reinforcing the assumption that the enzymes involved in the additions to exogenous oligosaccharides are the same as those involved in chondroitin polymerization. When either the pentasaccharide 6-sulfate or hexasaccharide 6-sulfate was incubated in reaction mixtures containing both of the sugar nucleotides there was generally growth of oligosaccharide by two or three sugars. With longer incubation under conditions of limiting oligosaccharide concentration, as many as 14 to 16 sugars could be added but no further chondroitin polymerization took place. Addition of each sugar was shown to depend upon the concentration of appropriate acceptor but was otherwise independent of the addition of the alternate sugar. No paired addition of sugars was noted. It was concluded that two specific enzymes are involved in alternate additions of sugars to the oligosaccharides and that the two enzymes have no apparent interaction with one another. It is suggested that the rapid polymerization to form large chondroitin chains which previously has been shown to take place on endogenous primers is facilitated by interaction of the two enzymes with a component of the endogenous primer. This component is not present in the exogenous oligosaccharides since they do not serve in the same fashion as primers for polymerization.

Acetylgalactosamine↗
PubMed1994

In vivo targeting function of N-linked oligosaccharides with terminating galactose and N-acetylgalactosamine residues.

N-Linked biantennary, triantennary, and core fucosylated biantennary oligosaccharides were isolated from animal glycoproteins and derivatized at their reducing end with Boc-tyrosine. The terminal Gal residues were enzymatically removed and replaced with GalNAc. Tyrosinamide-oligosaccharides were radioiodinated and administered intravenously to mice. Pharmacokinetic and biodistribution studies revealed structure-dependent differences in the steady-state volume of distribution, total body clearance rate, and targeting efficiency. Tyrosinamide-oligosaccharides were found to resist metabolism relative to a natural triantennary glycopeptide which was rapidly degraded in vivo. Triantennary oligosaccharides containing terminal Gal or Gal-NAc targeted the liver efficiently whereas biantennary oligosaccharides containing terminal Gal residues and differing only in their core fucosylation avoided recognition by the asialoglycoprotein receptor and were cleared unmetabolized by renal filtration. In contrast, biantennary oligosaccharides containing terminal Gal-NAc residues targeted the liver with much greater efficiency than Gal-terminated triantennary oligosaccharide. Core fucosylation reduced the metabolism rate of tyrosinamide-biantennary in the liver. The results establish the utility of tyrosinamide-oligosaccharides as probes to analyze the ligand specificity of mammalian lectins in vivo and demonstrate that a GalNAc-terminated biantennary is a potent ligand for the asialoglycoprotein receptor.

Acetylgalactosamine↗
PubMed1996

Elucidation of N-linked oligosaccharide structures of recombinant human factor VIII using fluorophore-assisted carbohydrate electrophoresis.

Characterization of the carbohydrate moiety is a critical measure of manufacturing process consistency of recombinant human Factor VIII (rFVIII) in Chinese-hamster ovary (CHO) cells. FVIII, a large (300 kDa) glycoprotein, is employed therapeutically for the correction of haemophilia A. While N-linked and O-linked oligosaccharides are found in this protein, the current study focuses on the N-linked oligosaccharides. The N-linked oligosaccharides from rFVIII were released using either peptide N-glycosidase F or endoglycosidase H, derivatized with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonate, and analysed by fluorophore-assisted carbohydrate electrophoresis (FACE). The electrophoretically resolved oligosaccharide bands were isolated and individual bands subjected to digestion with defined pools of exoglycosidases and re-electrophoresed on FACE sequencing gels. The resulting gel patterns were interpreted, based on band mobility shifts, to obtain the sequence structure of the oligosaccharides. A total of eight acidic and 12 neutral structures were identified, and the majority of the oligosaccharides (approximately 92%) were found to be sialylated. All of the major oligosaccharide structures found in CHO-cell-derived rFVIII have also been reported to be present in plasma-derived FVIII. Among the most abundant are disialylated, biantennary, core-fucosylated (approximately 40%), followed by trisialylated, triantennary, core-fucosylated and monosialo, biantennary, core-fucosylated structures (each approximately 18%). The Gal alpha 1-3Gal structures reported to be present in baby-hamster-kidney-cell-derived rFVIII were not found in the CHO-cell-derived protein. The glycosylation patterns were consistent in six random lots of rFVIII [coefficient of variation (%) 3-14] based on percentage lane luminance data of bands that represent approximately 98% of all asparagine-linked oligosaccharides.

Animals↗
PubMed1999

Effect of the labeling group in structural analyses of malononitrile-labeled oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization mass spectrometry

Structural analyses of oligosaccharide-malononitrile derivatives were conducted by matrix-assisted laser desorption/ionization post-source decay (MALDI-PSD) analysis in positive ion mode, and by electrospray ionization collision-induced dissociation (ESI-CID) analysis in negative ion mode. The malononitrile derivatives of oligosaccharides, which were developed for separation and detection of multi-component oligosaccharides by negative ion electrospray ionization mass spectrometry (ESI-MS), were detected by positive ion MALDI with a detection limit at a 400 fmol level even without any purification procedure for the derivatized oligosaccharide mixture. The results of structural analyses of oligosaccharide-malononitrile derivatives by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) were affected drastically by the choice of matrix used, and gave much more structural information than the free oligosaccharide. The effects of the malononitrile labeling group on the structural analyses of oligosaccharide-malononitrile derivatives by positive-ion MALDI-PSD analysis in reflectron mode, and by MALDI time-of-flight mass spectrometry (MALDI-TOFMS) in linear mode, are compared with those of in-source ESI-CID analysis in negative ion mode. The malononitrile derivatization method could be a powerful tool for structural analyses of oligosaccharides. Copyright 1999 John Wiley & Sons, Ltd.

Journal Article↗
PubMed1999

HIV infection and oligosaccharides: a novel approach to preventing HIV infection and the onset of AIDS.

Infection with human immunodeficiency virus type 1 (HIV) is triggered by binding of a glycoprotein called gp120 on the viral surface to CD4 molecules on the surface of target cells. Half of the gp120 glycoprotein is composed of oligosaccharides. It has been found that the gp120 oligosaccharides are essential in HIV infection and that high-mannose type oligosaccharides present in the gp120 molecule are especially critical. Investigation of gp120 oligosaccharides not only clarified the roles of oligosaccharides in HIV infection but also indicated a way to create novel anti-HIV agents by focusing on oligosaccharides. This review introduces the significance of oligosaccharides of the viral glycoprotein in HIV infection and our novel approach to preventing HIV infection and the onset of AIDS by targeting HIV oligosaccharides.

Journal Article↗
PubMed1996

Pectic polysaccharides from roots of Glycyrrhiza uralensis: possible contribution of neutral oligosaccharides in the galacturonase-resistant region to anti-complementary and mitogenic activities.

Digestion with endo-alpha-(1-->4)-polygalacturonase liberated the enzyme-resistant region (PG-1c) as an active site of the anti-complementary and mitogenic pectic polysaccharide (GR-2IIc) from Glycyrrhiza uralensis. Partial acid hydrolysis of PG-1c resulted in acidic oligosaccharides, and methylation analysis and GC-MS analysis of the acidic oligosaccharides suggested that PG-1c comprised a rhamnogalacturonan core such as -->2)-Rha-(1-->4)-GalA-(1-->2)-Rha-(1-->4)-GalA-(1-->-->4)-GalA-(1-->4) as the acidic moiety. Degradation of uronic acids by lithium decreased the anti-complementary and mitogenic activities of PG-1c. Although the products from PG-1c were still active, the methylglycoside of alpha-L-Rha-(1-->4)-alpha-D-GalA-(1-->2)-alpha-L-Rha-(1-->4)-alpha-D-Gal A did not show both activities. The products obtained by the lithium degradation from PG-1c gave fractions containing various neutral oligosaccharide-alditols. Among these fractions the longest and the short oligosaccharide-alditol fractions had relatively potent anti-complementary activity, whereas all oligosaccharide-alditol fractions expressed weak but significant mitogenic activity. GC-MS analysis indicated that the short oligosaccharide-alditol fraction contained various kinds of di- to tetrasaccharide-alditols. However, malto-oligosaccharide-alditols, and malto-, isomalto-, and laminari-oligosaccharides did not show anti-complementary and/or mitogenic activities, and these results suggested that certain neutral carbohydrate chains in PG-1c were responsible for the expression of mitogenic activity as well as anti-complementary activity of PG-1c.

Animals↗
PubMed1992

Circulatory half-life but not interaction with the lutropin/chorionic gonadotropin receptor is modulated by sulfation of bovine lutropin oligosaccharides.

Certain of the glycoprotein hormones, including bovine lutropin (bLH), bear asparagine-linked oligosaccharides terminating with the sequence SO4-4GalNAc beta 1-4GlcNAc beta 1-2Man alpha. To establish the biologic significance of these sulfate-bearing oligosaccharides we have compared properties of native bLH, desulfated bLH, recombinant bLH produced in Chinese hamster ovary cells that bears asparagine-linked oligosaccharides terminating with sialic acid alpha 2- 3Gal beta 1-4GlcNAc beta 1-2Man alpha rather than sulfated oligosaccharides (bLH/CHO), and desialyzed bLH/CHO. Using cultured MA-10 cells, a Leydig cell tumor line expressing the lutropin/chorionic gonadotropin receptor, we have found no differences in binding, cAMP production, or progesterone production between native and desulfated bLH. Sulfation of bLH oligosaccharides does not, therefore, modulate bLH bioactivity at the level of the lutropin/chorionic gonadotropin receptor. Removal of sulfate from bLH oligosaccharides and sialic acid from bLH/CHO oligosaccharides results in rapid clearance from the circulation by the hepatocyte asialoglycoprotein receptor. Thus sulfate, like sialic acid, prevents clearance from the circulation by the asialoglycoprotein receptor. The rapid removal of desulfated bLH from the circulation causes a 4- to 16-fold increase in the amount of bLH required to stimulate ovulation compared with native bLH. Particularly striking were differences in the metabolic clearance rates for native bLH and bLH/CHO, 7.3% per min and 1.7% per min, respectively. These differences were unexpected because bLH and bLH/CHO do not differ significantly in charge or size. The different metabolic clearance rates obtained for bLH and bLH/CHO indicate that the presence of sulfated rather than sialylated oligosaccharides on bLH results in a shorter circulatory half-life, which has a significant impact on in vivo bioactivity.

Animals↗
PubMed1980

Involvement of Lipid-linked Oligosaccharides in Synthesis of Storage Glycoproteins in Soybean Seeds.

Membrane preparations from developing soybean (var. Prize) cotyledon tissue, at the time of synthesis of storage glycoproteins, catalyze the sequential assembly of lipid-linked oligosaccharides from uridine-5'-diphospho-N-acetyl-d-[6-(3)H] glucosamine and guanosine-5'diphospho-d-[U-(14)C]mannose. The maximum size of lipid-linked oligosaccharide that accumulates contains the equivalent of 10 saccharide units on the basis of Bio-Gel P-2 gel filtration studies. These lipid-linked oligosaccharides show similar characteristics to polyisoprenyl diphosphate derivatives on diethylaminoethyl-cellulose chromatography and are potential intermediates in glycoprotein biosynthesis in this tissue. These glycolipids do not appear to turn over in pulse-chase experiments and no completed storage glycoproteins were detected among the products of these incubations.Tissue slices from cotyledons at the same stage of development synthesize lipid-linked oligosaccharides from [(3)H]mannose and [(3)H]glucosamine with sizes equivalent to 1, 7, 10, and approximately 15 saccharide units. In pulse-chase experiments, the lipid-linked saccharides with the equivalent of 1 and 10 units rapidly turnover, whereas those with 7 and 15 units do not. Examination of the higher oligosaccharide peaks (10 and 15) by Bio-Gel P-4 gel filtration shows them to comprise 2 distinct subsets of oligosaccharides containing different proportions of glucosamine and mannose units. Tissue slices synthesize products which resemble the completed 7S storage glycoproteins as judged by similarity of molecular weight and precipitation with specific antisera. Analysis of the oligosaccharides obtained by hydrazinolysis of glycoproteins shows the presence of a similar size "high-mannose" type N-linked oligosaccharides as in other glycoproteins from animal and plant cells.

Journal Article↗
PubMed1986

Oligosaccharide Side Chains of Glycoproteins that Remain in the High-Mannose Form Are Not Accessible to Glycosidases.

Glycoproteins present in the soluble and organelle fractions of developing bean (Phaseolus vulgaris) cotyledons were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, affinoblotting, fractionation on immobilized concanavalin A (ConA), and digestion of the oligosaccharide side chains with specific glycosidases before and after protein denaturation. These studies led to the following observations. (a) Bean cotyledons contain a large variety of glycoproteins that bind to ConA. Binding to ConA can be eliminated by prior digestion of denatured proteins with alpha-mannosidase or endoglycosidase H, indicating that binding to ConA is mediated by high-mannose oligosaccharide side chains. (b) Bean cotyledons contain a large variety of fucosylated glycoproteins which bind to ConA. Because fucose-containing oligosaccharide side chains do not bind to ConA, such proteins must have both high-mannose and modified oligosaccharides. (c) For all the glycoproteins examined except one, the high-mannose oligosaccharides on the undenatured proteins are accessible to ConA and partially accessible to jack bean alpha-mannosidase. (d) Treatment of the native proteins with alpha-mannosidase removes only 1 or 2 mannose residues from the high-mannose oligosaccharides. Similar treatments of sodium dodecyl sulfate-denatured or pronase-digested glycoproteins removes all alpha-mannose residues. The results support the following conclusions: certain side chains remain unmodified as high-mannose oligosaccharides even though the proteins to which they are attached pass through the Golgi apparatus, where other oligosaccharide chains are modified. The chains remain unmodified because they are not accessible to processing enzymes such as the Golgilocalized alpha-mannosidase.

Journal Article↗
PubMed1997

Rhizobium nodulation protein NodC is an important determinant of chitin oligosaccharide chain length in Nod factor biosynthesis.

Synthesis of chitin oligosaccharides by NodC is the first committed step in the biosynthesis of rhizobial lipochitin oligosaccharides (LCOs). The distribution of oligosaccharide chain lengths in LCOs differs between various Rhizobium species. We expressed the cloned nodC genes of Rhizobium meliloti, R. leguminosarum bv. viciae, and R. loti in Escherichia coli. The in vivo activities of the various NodC proteins differed with respect to the length of the major chitin oligosaccharide produced. The clearest difference was observed between strains with R. meliloti and R. loti NodC, producing chitintetraose and chitinpentaose, respectively. In vitro experiments, using UDP-[14C]GlcNAc as a precursor, show that this difference reflects intrinsic properties of these NodC proteins and that it is not influenced by the UDP-GlcNAc concentration. Analysis of oligosaccharide chain lengths in LCOs produced by a R. leguminosarum bv. viciae nodC mutant, expressing the three cloned nodC genes mentioned above, shows that the difference in oligosaccharide chain length in LCOs of R. meliloti and R. leguminosarum bv. viciae is due only to nodC. The exclusive production of LCOs which contain a chitinpentaose backbone by R. loti strains is not due to NodC but to end product selection by Nod proteins involved in further modification of the chitin oligosaccharide. These results indicate that nodC contributes to the host specificity of R. meliloti, a conclusion consistent with the results of several studies which have shown that the lengths of the oligosaccharide backbones of LCOs can strongly influence their activities on host plants.

Bacterial Proteins↗
PubMed1989

Alterations in the glycosylation of secreted thyrotropin during ontogenesis. Analysis of sialylated and sulfated oligosaccharides.

We have examined the carbohydrate structure of thyrotropin (TSH) secreted in vitro by pituitaries from prenatal, perinatal, and mature rats using concanavalin A (ConA)-agarose chromatography and anion-exchange high performance liquid chromatography (HPLC). [3H]Glucosamine-labeled TSH was immuno-precipitated and treated with either Pronase to generate glycopeptides or a mixture of endo-beta-N-acetyl-glucosaminidase F and peptide:N-glycosidase F to release oligosaccharides. The percentage of secreted TSH glycopeptides not bound to ConA was greater in mature animals (47 +/- 3%) than in either prenatal (29 +/- 3%) or perinatal animals (29 +/- 6%), suggesting more multiantennary oligosaccharides in the older animals. These structural changes were characterized further by performing anion-exchange HPLC on released oligosaccharides. Secreted TSH from prenatal rats predominantly contained oligosaccharides with 1 and 2 negative charges, while TSH from mature rats contained these structures as well as 15% with 3 negative charges. In addition, the ratio of sialylated to sulfated oligosaccharides was greater in mature compared to prenatal animals for species with 1 negative charge (1.9-fold) as well as for species with 2 negative charges (4.3-fold). We also correlated the structural alterations noted by ConA analysis with anion-exchange HPLC. Oligosaccharides that bound to ConA and were eluted with alpha-methylglucoside, when analyzed by anion-exchange HPLC, consisted of species with 1 and 2 negative charges, whereas oligosaccharides that were unbound to ConA were predominantly species with three negative charges. Together, these data suggest that with maturation of the hypothalamic-pituitary-thyroid axis secreted TSH contains more negatively charged multiantennary oligosaccharides with increased sialylation and decreased sulfation.

Age Factors↗
PubMed2026

Human Milk Oligosaccharides Modulate Nitrogen Utilization in Lactobacillus crispatus in a Glucose-Dependent Manner.

The vaginal microbiome's transition to a dysbiotic state increases susceptibility to pathogens like group B Streptococcus. While human milk oligosaccharides are established prebiotics in the neonatal gut, their impact on the vaginal niche remains largely unexplored. This study investigated the effects of pooled human milk oligosaccharides on the growth and physiology of vaginal (Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners) and gut-derived (Lactobacillus reuteri, Lactobacillus rhamnosus) commensals. Growth analyses revealed that human milk oligosaccharides significantly and selectively stimulated growth across all vaginal strains tested, whereas gut commensals exhibited variable or inhibited growth. Carbohydrate utilization assays and comparative genomics against Bifidobacterium infantis showed that Lactobacillus crispatus and Lactobacillus reuteri lack the canonical metabolic machinery to catabolize human milk oligosaccharides. Instead, nitrogen utilization assays identified a glucose-dependent pathway where human milk oligosaccharides are associated with the depletion of primary amines and amino acids in Lactobacillus crispatus supernatants. These results suggest that human milk oligosaccharides act as noncatabolic modulators of vaginal lactobacilli. Collectively, these in vitro findings may warrant investigation of human milk oligosaccharides as modulators of vaginal commensal physiology in more complex experimental systems.

Humans↗
PubMed1996

Oligomannosides or oligosaccharide-lipids as potential substrates for rat liver cytosolic alpha-D-mannosidase.

We have previously reported the substrate specificity of the cytosolic alpha-D-mannosidase purified from rat liver using Man9GlcNAc, i.e. Man alpha 1-2Man alpha 1-3(Man alpha 1-2Man alpha 1-6)Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3) Man beta 1-4G1cNAc, as substrate [Grard, Saint-Pol, Haeuw, Alonso, Wieruszeski, Strecker and Michalski (1994) Eur. J. Biochem. 223, 99-106]. Man9 G1cNAc is hydrolysed giving Man5GlcNAc, i.e. Man alpha 1-2 Man alpha 1-2Man alpha 1-3(Man alpha 1-6)Man beta 1-4GlcNAc, possessing the same structure as the oligosaccharide of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. We study here the activity of the purified cytosolic alpha-D-mannosidase towards the oligosaccharide-diphosphodolichol intermediates formed during the biosynthesis of N-glycans, and also towards soluble oligosaccharides released from the endoplasmic reticulum which are glucosylated or not and possessing at their reducing end either a single N-acetylglucosamine residue or a di-N-acetylchitobiose sequence. We demonstrate that (1) dolichol pyrophosphate oligosaccharide substrates are poorly hydrolysed by the cytosolic alpha-D-mannosidase; (2) oligosaccharides with a terminal reducing di-N-acetylchitobiose sequence are not hydrolysed at all; (3) soluble oligosaccharides bearing a single reducing N-acetylglucosamine are the real substrates for the enzyme. These results suggest a role for alpha-D-mannosidase in the catabolism of glycans released from the endoplasmic reticulum rather than in the regulation of the biosynthesis of asparagine-linked oligosaccharides.

Animals↗
PubMed1988

Characterization of an oligosaccharide-pyrophosphodolichol pyrophosphatase activity in yeast.

Use of Triton X-114 allowed us to develop a new method to separate hydrophilic oligosaccharidic material from hydrophobic oligosaccharide pyrophosphodolichols (oligosaccharide-PP-Dol). Taking advantage of this procedure we characterize, in yeast microsomal membranes, an enzymic activity that hydrolyses oligosaccharide-PP-Dol into oligosaccharidic material. H.p.l.c. analysis together with alkaline-phosphatase- and endo-N-acetyl-beta-D-glucosaminidase-susceptibility demonstrate that the oligosaccharidic released material is mainly composed of oligomannosides containing a chitobiose phosphate at the reducing end. The enzymic activity requires bivalent cations and is inhibited by pyrophosphate, NAD+ and bacitracin. As other, commercially available, pyrophosphatases have no action on lipid intermediates, the described pyrophosphatase activity appears to be the specific enzyme for oligosaccharide-PP-Dol. This enzymic splitting of the pyrophosphate bond might be the primary event in the catabolism of lipid intermediates.

Bacitracin↗
PubMed1997

Characterization of oligosaccharide composition and structure by quadrupole ion trap mass spectrometry.

The use of electrospray ionization-quadrupole ion trap mass spectrometry for the characterization of linear oligosaccharides and N-linked protein oligosaccharide mixtures is described. Tandem mass spectrometry (MS/MS) experiments with orders higher than two offer a number of ways to enhance MS/MS spectra and to derive information not present in MS and MS2 spectra. Three such methods are presented in this paper. (a) Collisional activation of permethylated oligosaccharide molecular ions (MS2) as illustrated by maltoheptaose, produces abundant fragments from glycosidic bond cleavages which indicate composition and sequence, and weak cross-ring cleavage products which denote specific linkages within the oligosaccharide. Through the trapping and further dissociation of these fragments (MSn), cross-ring cleavage products can be confirmed and their relative abundances increased to facilitate interpretation. (b) The mechanisms of formation of two isobaric ions or ions isobaric with another ion's isotope peaks, such as those present in the MS2 spectrum of the ribonuclease B oligosaccharide GlcNAc2-Man5 can be independently established by separate MS3 experiments. (c) Ions in the MS2 spectrum, specific for individual components of an isobaric mixture, can be isolated and characterized by further stages of fragmentation. This is illustrated by two isobaric oligosaccharides from chicken ovalbumin of the composition HexNAc5Hex5. These findings indicate the utility of ion trap mass spectrometry towards the facile determination of oligosaccharide composition, sequence, branching and linkage, providing a wealth of structural information not obtainable by other individual methods of carbohydrate mass spectrometric analysis.

Carbohydrate Sequence↗
PubMed1998

Detection of low-molecular-weight heparin oligosaccharides (Fragmin) using surface plasmon resonance.

During the last decades there has been a growing realization of the central biological role that oligosaccharides and oligosaccharide-protein interactions play. One of the most striking examples is the use of heparin and low-molecular-weight heparin oligosaccharides (Fragmin) to modify blood coagulation. Several monoclonal antibodies directed against glycosaminoglycan structures have been produced. However, their clinical use is limited by the difficulty of detection systems for oligosaccharides. In the present study we used a monoclonal antibody directed against heparin oligosaccharides prepared by partial nitrous acid deamination of heparin. Using a biosensor (BIAcore), purified antibody was immobilized on sensor surfaces and binding of oligosaccharide was measured by surface plasmon resonance. Using this technique, it was possible to quantitate low-molecular-weight heparin oligosaccharides in nanomolar concentrations.

Animals↗
PubMed1989

Carbohydrate recognition in neuronal development: structure and expression of surface oligosaccharides and beta-galactoside-binding lectins.

The differentiation and development of vertebrate neurons is controlled in part by interactions with cell surface and extracellular matrix molecules, many of which are glycoproteins that mediate their developmental actions by homophilic or heterophilic binding to other glycoproteins. In addition there is increasing evidence that cell recognition and adhesion in some embryonic cell types involve interactions between cell surface oligosaccharides and complementary carbohydrate-binding proteins. Although a role for carbohydrate recognition in neuronal development has been proposed, the precise function of complex carbohydrate structures on neural cells has not been defined. To approach this problem, we have examined the structure and expression of cell surface oligosaccharides and carbohydrate-binding proteins by primary sensory neurons in the rat dorsal root ganglion (DRG). There are several functionally distinct subsets of DRG neurons, each of which conveys a different sensory modality to distinct target domains in the spinal cord. Monoclonal antibodies against defined oligosaccharide structures identify each of the major subsets of DRG neurons on the basis of their expression of a distinct set of complex oligosaccharides, derived from lacto-, globo- and ganglioseries backbone structures. In particular, small diameter DRG neurons involved in pain processing express beta-galactoside-based lactoseries oligosaccharides. DRG and spinal cord neurons also express two soluble beta-galactoside-binding proteins of relative molecular masses 14,500 and 29,000, termed RL-14.5 and RL-29, which represent potential ligands for lactoseries oligosaccharides. RL-14.5 is expressed by the majority of DRG neurons whereas RL-29 is restricted to the subset of small DRG neurons that express surface N-acetyllactosamine structures. RL-14.5 and RL-29 are expressed soon after the differentiation of DRG neurons and appear to be released from cultured DRG neurons. Rat brain cDNA clones encoding RL-14.5 have been isolated. The nucleotide and predicted amino acid sequence of RL-14.5 has confirmed that this lectin is highly homologous to soluble beta-galactoside-binding proteins in other vertebrate species. Northern blot analysis and in situ hybridization indicate that RL-14.5 mRNA is selectively expressed in sensory and motor neurons in the rat nervous system. The selective expression of lactoseries oligosaccharides and complementary beta-galactoside-binding lectins may contribute to the differentiation and/or development of these two classes of neurons.

Aging↗